Project Summary In this application we address the need for influenza drugs with novel targets, and specifically we propose to discover antiviral compounds that target the influenza virus polymerase complex. Currently there is only one class of influenza drug that is clinically useful; the neuraminidase inhibitors. Relying on a single drug class is problematic as the emergence of resistant virus could render these drugs ineffective as has already happened with the adamantane drugs targeting the viral M2 protein. In fact, concerns about resistance to oseltamivir, an oral neuraminidase inhibitor that is the most widely prescribed influenza drug, are driving the call for influenza drugs with new targets and mechanisms. The viral polymerase is an essential catalytic component of all viruses, and has proven to be an excellent target for development of potent antiviral drugs, but as yet no polymerase inhibitors have been approved for influenza treatment. In preliminary studies we have performed a high-throughput screen of a 900,000 compound library using a cell-based assay and identified 744 hits with influenza antiviral activity. We propose to use a novel in vitro influenza polymerase assay to rapidly identify polymerase inhibitors amongst these validated hits. This unique assay captures all the essential steps leading to formation of the final RNA product and thus will allow us to identify novel compounds that inhibit endonuclease activity, mRNA cap-binding, RNA-binding, and RNA synthesis. Specific assays will be employed to elucidate the antiviral mechanism and we will also perform studies to select for resistance as a means to identify the target and determine the barrier to resistance. Our overall goal is to identify one or more influenza virus polymerase inhibitors that are potent and display antiviral breadth across multiple influenza A virus subtypes and influenza B virus. Such compounds present an exciting starting point for antiviral development but will also serve as probes in ongoing efforts to define influenza virus polymerase function.